PROJECT SUMMARY (See instructions); We are exploring the hypothesis that the Corticotropin Releasing Factor (CRF)/urocortin family of ligands and their cognate receptors play specific and critical roles in the modulation of adaptive responses to stress and other circumstances. In previous granting periods, this Project, with collaborators throughout the Program, characterized CRF, cloned 2 CRF receptors, CRFR1 and CRFR2 and identified 3 urocortins (Ucn 1, 2 & 3) and cloned CRF-BP, a bio-neutralizing binding protein. Ucn 2 and 3 precursors were deduced from DNA databases. Because their mature forms have not been identified, we will isolate and sequence the peptides from tissues and cell secretions. Our receptor studies have focused on exploring the roles of the N-terminal, first extracellular domain (ECD1) of the CRFRs, which include critical binding sites for peptide agonists and antagonists. We will continue to collaborate with Project 3, to solve the structures of additional ligand-bound ECD1 of CRF receptors by NMR and will explore means of expressing and evaluating functional full-length receptors. Mice deficient in each of the 3 urocortins developed in collaboration with Core B have been characterized. We have been instructed by the metabolic phenotypes of receptor and ligand null mice as well as by the anatomic distributions of CRFR2, Ucn 2 and Ucn 3. We proposed that Ucn 2, strongly expressed in skeletal muscle, plays a local role within that tissue to negatively regulate insulin sensitivity. Whereas, Ucn 3 found in beta cells of the pancreatic islet, enhances glucose stimulated insulin secretion. Because of possible interest in increasing insulin sensitivity by selectively blocking Ucn 2, we are developing mutant CRF-BPs that bind and neutralize Ucn 2 but display minimal affinities for CRF, Ucn 1 and Ucn 3, thereby avoiding suppression of glucocorticoid production and possible untoward effects on islet, heart and other tissues. We have also explored the role of CRF and CRFR1 in the islet and found this receptor/ligand system to also enhance glucose-mediated insulin secretion. We propose to fully define the distributions of ligands and receptors of the CRF network within the islet and to explore the regulation of the CRFR1 and CRFR2 components and cross-talk between the two receptor systems. Finally, we will examine this system in models of obesity. Our goal is to understand the physiologic context of the CRF/urocortin network and to reveal potential means of exploiting this information for the management of human stress-related and metabolic diseases.